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- EMDB-8477: CRISPR RNA-guided surveillance complex -

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Basic information

Entry
Database: EMDB / ID: EMD-8477
TitleCRISPR RNA-guided surveillance complex
Map data
Sample
  • Complex: CRISPR RNA-guided surveillance complex
    • Protein or peptide: CRISPR-associated protein, Cse3 family
    • Protein or peptide: Cse2Carbon diselenide
    • Protein or peptide: CRISPR-associated protein, Cse1 family
    • Protein or peptide: CRISPR-associated protein, Cse4 family
    • RNA: crRNA
    • DNA: Target Strand
    • Protein or peptide: CRISPR-associated protein, Cas5e family
    • DNA: Nontarget Strand
KeywordsCRISPR-Cas / Cascade / IMMUNE SYSTEM
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / defense response to virus / RNA binding / identical protein binding
Similarity search - Function
CRISPR-associated protein, CT1975 / CT1975-like protein / CRISPR-associated protein, CasD / CRISPR-associated protein Cse2 / Cse2 superfamily / CRISPR-associated protein Cse2 (CRISPR_cse2) / CRISPR-associated protein Cse1 / CRISPR-associated protein Cse1 (CRISPR_cse1) / CRISPR-associated protein Cse3 / CRISPR associated protein ...CRISPR-associated protein, CT1975 / CT1975-like protein / CRISPR-associated protein, CasD / CRISPR-associated protein Cse2 / Cse2 superfamily / CRISPR-associated protein Cse2 (CRISPR_cse2) / CRISPR-associated protein Cse1 / CRISPR-associated protein Cse1 (CRISPR_cse1) / CRISPR-associated protein Cse3 / CRISPR associated protein / CRISPR_assoc / CRISPR-associated protein, Cas5 / CRISPR-associated protein (Cas_Cas5) / CRISPR-associated protein Cas5, N-terminal
Similarity search - Domain/homology
CRISPR-associated protein, Cse1 family / CRISPR-associated protein, Cse2 family / CRISPR-associated protein, Cse4 family / CRISPR-associated protein, Cas5e family / CRISPR-associated protein, Cse3 family
Similarity search - Component
Biological speciesThermobifida fusca (strain YX) (bacteria) / Thermobifida fusca YX (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsXiao Y / Luo M
CitationJournal: Cell / Year: 2017
Title: Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System.
Authors: Yibei Xiao / Min Luo / Robert P Hayes / Jonathan Kim / Sherwin Ng / Fang Ding / Maofu Liao / Ailong Ke /
Abstract: Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two cryo- ...Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two cryo-EM snapshots of the Thermobifida fusca type I-E Cascade: (1) unwinding 11 bp of dsDNA at the seed-sequence region to scout for sequence complementarity, and (2) further unwinding of the entire protospacer to form a full R-loop. These structures provide the much-needed temporal and spatial resolution to resolve key mechanistic steps leading to Cas3 recruitment. In the early steps, PAM recognition causes severe DNA bending, leading to spontaneous DNA unwinding to form a seed-bubble. The full R-loop formation triggers conformational changes in Cascade, licensing Cas3 to bind. The same process also generates a bulge in the non-target DNA strand, enabling its handover to Cas3 for cleavage. The combination of both negative and positive checkpoints ensures stringent yet efficient target degradation in type I CRISPR-Cas systems.
History
DepositionNov 23, 2016-
Header (metadata) releaseMar 22, 2017-
Map releaseAug 9, 2017-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5u07
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8477.png

Downloads & links

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Map

FileDownload / File: emd_8477.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.23 Å
Density
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.045
Minimum - Maximum-0.107699394 - 0.21361065
Average (Standard dev.)0.000027352522 (±0.006874965)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 314.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.231.231.23
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z314.880314.880314.880
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-26-36
NX/NY/NZ528549
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1080.2140.000

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Supplemental data

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Sample components

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Entire : CRISPR RNA-guided surveillance complex

EntireName: CRISPR RNA-guided surveillance complex
Components
  • Complex: CRISPR RNA-guided surveillance complex
    • Protein or peptide: CRISPR-associated protein, Cse3 family
    • Protein or peptide: Cse2Carbon diselenide
    • Protein or peptide: CRISPR-associated protein, Cse1 family
    • Protein or peptide: CRISPR-associated protein, Cse4 family
    • RNA: crRNA
    • DNA: Target Strand
    • Protein or peptide: CRISPR-associated protein, Cas5e family
    • DNA: Nontarget Strand

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Supramolecule #1: CRISPR RNA-guided surveillance complex

SupramoleculeName: CRISPR RNA-guided surveillance complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Source (natural)Organism: Thermobifida fusca (strain YX) (bacteria)

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Macromolecule #1: CRISPR-associated protein, Cse3 family

MacromoleculeName: CRISPR-associated protein, Cse3 family / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermobifida fusca YX (bacteria) / Strain: YX
Molecular weightTheoretical: 26.327938 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTWLTKIVPD LRYRQTRADF RTAGNLHRKL IRLSSDLGEE RIANPRQQSG LLFRIEETRN ELYLLVQSHS PLRVDRLGPG YHGVQMRNL DPFLARLDKG SRVRYRIVAS PTKRLGRSEN NTQRLGLKEP PKKPREYTWA LRGAAAEEWW HSRAAANGLE L LSTYAQTL ...String:
MTWLTKIVPD LRYRQTRADF RTAGNLHRKL IRLSSDLGEE RIANPRQQSG LLFRIEETRN ELYLLVQSHS PLRVDRLGPG YHGVQMRNL DPFLARLDKG SRVRYRIVAS PTKRLGRSEN NTQRLGLKEP PKKPREYTWA LRGAAAEEWW HSRAAANGLE L LSTYAQTL DDVRDPGTAD RSRKIRHPAV RFDGEAVISD VDAVRHAVLN GIGRGKSYGC GLLSLALIEE GEHG

UniProtKB: CRISPR-associated protein, Cse3 family

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Macromolecule #2: Cse2

MacromoleculeName: Cse2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Thermobifida fusca YX (bacteria) / Strain: YX
Molecular weightTheoretical: 27.446613 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNSDYILQHA DALVKRVSKL IVNEPAARAA LRRGVGLAPE DPRMLAAHRV VAPYVPVPTD YDVDRRRAAS LWDVHAVERA FYAVAAIMA AQPRSARDQE AEATEEQTGE PQDSEALTEP TPAEESSATK DGKPDRRPNL GVSLAQAVFD KGLNADSTEQ R LHLIARQN ...String:
MNSDYILQHA DALVKRVSKL IVNEPAARAA LRRGVGLAPE DPRMLAAHRV VAPYVPVPTD YDVDRRRAAS LWDVHAVERA FYAVAAIMA AQPRSARDQE AEATEEQTGE PQDSEALTEP TPAEESSATK DGKPDRRPNL GVSLAQAVFD KGLNADSTEQ R LHLIARQN LDGVHRHLPR LVLYLRSDQV HIDWGILIRD LARWGHTPRH VAREWVQDYH RTLETLTRQA EQKNKNNTTD EE AEAA

UniProtKB: CRISPR-associated protein, Cse2 family

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Macromolecule #3: CRISPR-associated protein, Cse1 family

MacromoleculeName: CRISPR-associated protein, Cse1 family / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermobifida fusca YX (bacteria) / Strain: YX
Molecular weightTheoretical: 61.433297 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MLSVALCFLV GGAIPSPPSF DVTIAPWLIA RSRDVLAAPE MLGLRDVLIR SHELSDVEIP LPPGAAVLWR ILALITARIT GLDQPPNKN PKRKWQARRS QILSKGRLDP EAVDAYFADY SERFDLFHPE RPWLQDPRLR EECPKTSGVN KLAWGRTAGE N QVWLGGHH ...String:
MLSVALCFLV GGAIPSPPSF DVTIAPWLIA RSRDVLAAPE MLGLRDVLIR SHELSDVEIP LPPGAAVLWR ILALITARIT GLDQPPNKN PKRKWQARRS QILSKGRLDP EAVDAYFADY SERFDLFHPE RPWLQDPRLR EECPKTSGVN KLAWGRTAGE N QVWLGGHH HDLDPHPLDS AEAVWHLLAT LGYGPSGMCT ARVVRGRSER NVTAGPLRGT VSYHPLGRTL FESLILNIPY PG TGAADLA FWEQPELNDP LGLPEESAGL AGILRLDHFR HAVLLHPSPD GSHVVDAWVT WAWRERNISP ELDPYLIYQT SKE GRVYPR PAEAERAIWR DLDALLHYGE DGNYRPTILD NCTPLAQVPQ EVLDSLRLRA FGFDQDGQAR DKQWFTATTP AVLR WLADR ETDDNENARI VRRITLARKA AEALGRRLEK ACKEAWKESN SPSSTSSGTN AKTETGVGPW VQHGMSRYWA KAEPV FWNI VYDRPAQGYT PGMAGPGNAF NLVALAAYDE VTGPYCERPR VAKVVERHRS TLFSNWTPKQ DKEAA

UniProtKB: CRISPR-associated protein, Cse1 family

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Macromolecule #4: CRISPR-associated protein, Cse4 family

MacromoleculeName: CRISPR-associated protein, Cse4 family / type: protein_or_peptide / ID: 4 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Thermobifida fusca YX (bacteria) / Strain: YX
Molecular weightTheoretical: 41.043043 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTFVDIHAIQ TLPYSNINRD DLGSPKTVVY GGKERTRVSS QSWKRAVRHE VEARLGDKAV RTRRIISEIA KRLRERGWDA DLADAGARQ VVLSVGKKSG IKLEKEKDSE APATSVLFYL PVPAIDELAA IADEHRDAVA KEAAKKTPKG ILPADRITEV L KSRNVSVN ...String:
MTFVDIHAIQ TLPYSNINRD DLGSPKTVVY GGKERTRVSS QSWKRAVRHE VEARLGDKAV RTRRIISEIA KRLRERGWDA DLADAGARQ VVLSVGKKSG IKLEKEKDSE APATSVLFYL PVPAIDELAA IADEHRDAVA KEAAKKTPKG ILPADRITEV L KSRNVSVN LFGRMLAELP STEVDGAVQF AHAFTVHGTT VEVDFFTAVD DIPKENDHGS GHMNAGQFSA GTFYRYANVN LD RLVENTG DAQTARTAVA EFLRAFLSTV PSGKQNATAA MTLPDLVHIA VRFDRPISFA PAFETALYGS DGYTLRACQE LNN YAERLR EVWPDDAIRG YATVENKTDL AALGERYDSY PALIDAMVAA AFEGERE

UniProtKB: CRISPR-associated protein, Cse4 family

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Macromolecule #7: CRISPR-associated protein, Cas5e family

MacromoleculeName: CRISPR-associated protein, Cas5e family / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermobifida fusca YX (bacteria) / Strain: YX
Molecular weightTheoretical: 28.27926 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSGFLLRLAG PMQSWGEHSM FGERDTLPYP SRSGLIGMFA AAQGVRRGDP LDRYKELKFT VRVDRPGVRL VDFHTIGGGL PKERTVPTA AGERRDPKKA TIVTSRSYLA DAVFTVAVTG PEADTIADAL AAPYWQPYLG RRAFVPDPLL VLRRRVADPV R ELVEAVPL ...String:
MSGFLLRLAG PMQSWGEHSM FGERDTLPYP SRSGLIGMFA AAQGVRRGDP LDRYKELKFT VRVDRPGVRL VDFHTIGGGL PKERTVPTA AGERRDPKKA TIVTSRSYLA DAVFTVAVTG PEADTIADAL AAPYWQPYLG RRAFVPDPLL VLRRRVADPV R ELVEAVPL PHRRVEEDAA TVLVDLIYEE GEYPDTRTLT VLNDVPLSFD SKSRRYSTRQ IRVVPTEVPA TLVAGPGRDY QN KLFTYVK QCAEEAA

UniProtKB: CRISPR-associated protein, Cas5e family

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Macromolecule #5: crRNA

MacromoleculeName: crRNA / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Thermobifida fusca YX (bacteria)
Molecular weightTheoretical: 19.790793 KDa
SequenceString:
AUGGACCGCC AGUGAUAAGU GGAAUGCCAU GUGGGCUGUC GUGAGCCCCA CGCACGUGGG G

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Macromolecule #6: Target Strand

MacromoleculeName: Target Strand / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Thermobifida fusca YX (bacteria)
Molecular weightTheoretical: 6.381106 KDa
SequenceString:
(DT)(DT)(DA)(DT)(DC)(DA)(DC)(DT)(DG)(DG) (DC)(DT)(DT)(DC)(DG)(DT)(DC)(DC)(DG)(DC) (DG)

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Macromolecule #8: Nontarget Strand

MacromoleculeName: Nontarget Strand / type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Thermobifida fusca YX (bacteria)
Molecular weightTheoretical: 3.9866 KDa
SequenceString:
(DC)(DG)(DC)(DG)(DG)(DA)(DC)(DG)(DA)(DA) (DG)(DC)(DC)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.8 mg/mL
BufferpH: 7.5 / Details: 10 mM Hepes, 150 mM NaCl, 5 mM DTT, pH 7.5
GridModel: 400 mesh Quantifoil holey carbon grid / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 85 %

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.3000000000000003 µm / Calibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal magnification: 31000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN / Cooling holder cryogen: NITROGEN
TemperatureMin: 80.0 K / Max: 105.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 1305 / Average electron dose: 8.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 582497
Startup modelType of model: NONE
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: SAMUEL
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 70222

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